Fetal Alcohol Spectrum Disorder (FASD)

Facial Features of Children with FASD
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Fig1. Adapted from Watterndorf et al. [2005][14]. Distinctive facial feature in children from different cultural backgrounds with FASD.

Fetal Alcohol Spectrum Disorder (FASD) is an umbrella term which encompasses a continuum of birth defects derived from prenatal alcohol exposure caused by maternal alcohol consumption during pregnancy. The term itself is not a clinical diagnosis of a disorder, but includes the diagnostic conditions of alcohol-related neurodevelopmental disorder (ARND), fetal alcohol syndrome (FAS), partial FAS, and alcohol-related birth defects (ARBD). The diagnoses for the diagnostic conditions of FASD make use of physical and neurobehavioural examinations, and maternal alcohol consumption history. The symptoms can include, but are not limited to growth retardation, musculoskeletal disabilities, cognitive deficits in learning and memory, attention, and intellectual performance1. Additionally, individuals with FASD present a higher risk for developing behavioural issues which include psychiatric disorders, difficulty with social interactions, and substance abuse1. Although many symptoms have been identified, little is known about the underlying mechanism of the pathogenesis. However, it is understood that microglia, the resident macrophage of the central nervous system (CNS), is a target of ethanol pathogenesis and adopts lasting morphological changes resulting from CNS-wide inflammation1. Furthermore, emerging evidence suggest a Toll-like receptor 4 (TLR4) mediated inflammatory response by microglia and a reduction in histone acetylation across numerous brain regions emerging from prenatal alcohol exposure1. Despite the major advances in medical diagnosis, there is no treatment for FASD. Nevertheless, PPAR-γ agonists, a group of immunosuppressive drugs, have recently been shown to protect microglia and neurons by reducing the alcohol-induced inflammation in mouse models2. Early intervention services have also demonstrated to be effective in improving a child’s development1.

1.1 Diagnosis

1.1a Screening and Physical Examination

Screening for the diagnostic conditions of FASD begins with a dysmorphology assessment of the body to distinguish physical features that are abnormally present and related to the characteristics of prenatal alcohol exposure. An ordinary physical examination of the head is required to discern characterizing features of FASD which includes, but not limited to a highly arched palate, hypertelorism, unaligned teeth, a short vermillion border, a flat philtrum, and shortened palpebral fissures. Additionally, regular screenings are used in conjunction to an initial physical examination to detect signs of growth deficiency, such as a considerably low weight or height and a small head circumference, a determining factor of FASD. In case of children with dysmorphic features as a result of other genetic disorders, a genetic screening is recommended for further confirmation3.

1.1b Neurobehavioral assessment

In conjunction to a physical examination required for identifying FASD, a cognitive and behavioural assessment is necessary for proper diagnosis. Although the cognitive and behavioural outcomes of FASD may differ from case to case, it typically affects an individual’s memory, executive function, attention, social skills, cognition, and sensory and motor skills. Within each category, basic and advance exercises are used for assessment, while three categories demonstrating signs of impairment is a minimum requirement for diagnosis. Furthermore, brain structure abnormality is screened using magnetic resonance imaging (MRI) for additional confirmation and correlation to its associated behavioural deficits expressed in the patient. In cases where standardised assessments are lacking, age, socioeconomic, mental health and environment are taken into account as they may impact development without signs of brain injury3.

1.1c Criteria for FAS, partial FAS and ARND

The 4 Digit Diagnostic Code
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Table1. Adapted from Chudley et al.[2005][3] An illustration of the 4 Digit Diagnostic Code criteria for FASD used by clinicians.

The diagnostic processes of FAS, pFAS, ARBD, and ARND rely on physical examinations and neurobehavioural assessments which can be based on several evaluation methods. This includes the 4-digit code (Table1), the Center for Disease Control (CDC) method, the Modified Institute of Medicine method (IOM), and the Canadian guideline for FASD diagnoses which consists a combination of the IOM and the 4-digit code method. Although several methods of diagnosis exist, each one evaluates the evidence base on the same criterion: growth deficiency, FASD facial features, central nervous system dysfunction, and prenatal exposure to alcohol. The evidence from the four domains is rated on the severity of expression which will determine its diagnostic condition. However, the disparity among methods is based on the how each diagnostic condition is defined, and can lead to different diagnoses between methods for the same individual3.

1.1d Risk factors

In terms of risk factors, studies suggest that prenatal alcohol exposure occurs mostly among older mothers with lower education levels and socioeconomic class, and paternal alcohol and drug consumption during maternal pregnancy. Additionally, parental smoking habits and cocaine abuse have also shown to be related to high incidence of FASD, as well as poor nourishment, and exposure to mentally and physically stressful environments. Other studies, including a cohort study of five years, determined that mothers with children diagnosed with FASD arrived from a varied racial and socioeconomic background, contradicting with previously known risk factors (Figure 1). However, incidences were often associated with sexual abuse and social isolation. Overall, there are insufficient studies on FASD and its related risk factors; therefore are no numbers to accurately predict risk levels. Nonetheless, the greatest risk is directly related to prenatal alcohol exposure in a dose-dependent manner3.

1.2 Alcohol-Induced CNS Inflammation

Microglia Migration
Vid1. Adaptive from Costandi.[2012] A illustration of the amoeboid-like characteristics of activated microglia.

Chronic alcohol consumption and its effects on the human immune system have not yet been well characterized, however, it has been accepted that its effects on the body can lead to an immunosuppressed state and promote the development of infection as well as liver, and other forms of inflammation4. Alcohol consumption has also led to many cases of CNS inflammation whereby the resulting effects left individuals with abnormal cognitive functions and a degenerating nervous system1. In the case of FASD, alcohol exposure to neurons, microglia, and astrocytes have been the focus of investigation, however, only recently the effects of microglial, the resident phagocytic cell in the CNS, has become the main focus of research.

1.2a Microglia Activation and Altered Morphology

In the CNS, the biological role of microglia involves promoting homeostasis; producing chemokines, releasing neurotrophic factors, and the removal of harmful products by way of phagocytosis for the purpose of defending other resident cells. This phagocytic cell, following activation, can induce the production of pro-inflammatory molecules such as cytokines and nitrogen reactive species. Conjointly, microglia play a critical role regulating hormones, neurotransmitters, and act as modulators in the CNS. By way of growth factor secretion, microglial cells also induce the survival of resident CNS cells and are involved in synaptic pruning, which are both equally essential for proper CNS development. Despite their protective role in the CNS, recent studies have associated chronically activated microglia with neuronal and glial cell death in which the phenotype were reminiscent of other neuro-inflammatory disorders. When triggered by cell injury, infections, or cytokines, the microglia cells develop an altered morphology resembling an amoeboid state, and activates the release of inflammation inducing molecules including TNF-a, IL-1B and MCP-1. During this activated state, its amoeboid morphology (Video 1) allows for greater motility and antigen presenting capacity for T cells activation that may also contribute to a cascade of immune cell activation and further CNS inflammation(Video 1). Once the inflammatory inducing environment is cleared, microglial cells will revert to its original morphology, however, recent studies demonstrate its susceptibility for future activation1.

1.2b Alcohol Consumption and Inflammation in Humans

Individuals exhibiting chronic alcohol consumption consistently bear high levels of pro-inflammatory molecules including TNF-a, IL-1B and IL-65. Additionally, alcohol consumption has been shown to induce the production of pro-inflammatory molecule by blood monocyte in which excessive amounts can lead to liver inflammation6. By examining gene expression in post-mortem brain from chronic alcohol consumers, high levels of NF-kB was determined in addition to other pro-inflammatory cytokines6. This suggests that alcohol consumption may contribute to the behavioural impairment seen in FASD due to high levels of NF-kB and its involvement in synaptic plasticity7. Furthermore, transgenic animal experiments associated with excessive MCP-1 expression modified hippocampus synaptic transmission, which in addition to NF-kB, may also contribute to cognitive deficits1. Moreover, TNF-a, IL-1B and IL-6 showed elevated expression in both fetal and maternal blood in in chronic alcohol consumers, indicating that maternal ingestion of alcohol clearly elevates fetal cytokine levels as well8. The study, however, did not categorise the source of expression as either maternal or fetal, but confirmed its presence in the fetus. Nevertheless, fetal exposure to alcohol resulted in elevated cytokines levels and induced similar alcohol derived phenotypes seen in the mother8.

1.2c Alcohol Consumption and Inflammation in Animal Models

An Equivalent Comparison of Dysmorphology in rodents and humans
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Fig2. Adapted from Mattson et al.[1994][15]An example of comparable corpus callosum dymorphology in rodents and humans.

Mouse models involving prenatal, postnatal and neonatal alcohol exposure are common experimental models of FASD1 (Figure 2). Mouse models are similar to humans in that CNS development during the third trimester in humans reflects an equivalent stage in a neonatal mouse brain9. Studies determined that neonatal exposure to alcohol in mice resulted in fewer and abnormal morphology of microglia in the cerebellum1. Furthermore, this altered morphology suggested a partial activation of microglia that may detrimentally modify the developmental process of the CNS1. Since microglial function is essential to neuronal and glial health, an altered microglial state may result in a persistent change of states that will consequently affect its function throughout development1. Similarly, pro-inflammatory cytokine expression lasting twenty day after withdrawal was also observed, reflecting the altered microglial state. Therefore, it is hypothesized that modifications to the developing neuro-immune system promote lasting pathological effects on the CNS1.

In addition to altered microglia morphology, high levels of oxidative stress in the CNS during development have been shown to increase the rate of neuronal and glial cell loss1. Superoxide dismutase, lipid peroxidase, nitric oxide and other indicators of oxidative stress demonstrated high levels of expression in the cerebral cortex and hippocampal areas, and was also co-expressed with pro-inflammatory cytokines and caspase-3, an inducer of programmed cell death1. This suggested that oxidative stress performed an important part in alcohol induced pathology. Furthermore, alcohol has been known to be toxic to neurons and glial cells10. Direct exposure to alcohol in microglia-neuron co-cultures increased the rate of neuronal cell loss by way of TNF-a mediated apoptosis, indicating that primed microglia promotes programmed cell death10. The neurons involved in the co-culture include cortical, Purkinje, and hippocampal neurons, suggesting their vulnerability during alcohol exposure such as in the case of FASD, where they may reflect cognitive function, memory and motor tasks deficiency, respectively1.

1.2d Microglia-Dependant TLR-4 Signalling Pathway Mediating Inflammation

PRRs Involved in Pro-inflammatory Gene Expression
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Fig3. Adapted from Block et al.[2007][16]An overview of the mechanisms involved in pro-inflammatory gene expression induced by TLR4 and other PRRs.

Generally, microglia activation due to pathogen infection occurs through TLRs mediated mechanisms, however, emerging evidence suggest that a TLR4 dependant inflammatory response is also mediated by microglial cells1(Figure 3). This has been shown using wild type (WT) mice in which alcohol exposure induced cytokine production and apoptosis through a signal transduction pathway consisting of p38 MAP, ERK and JNK1. Since TLR4 deficient mice lacked the ability to do either, the study suggests that TLR4 has a contributing role in CNS inflammation and neuronal survivability1. Additionally, the study demonstrated that the expression of caspase-3, a marker of apoptosis, was mediated through a TLR4-dependant mechanism indicating the role of TLR4 in neurodegeneration1. Upon activation, TLR4 can also induce a MyD88-dependant or independent pathway; however few studies have attempted to address this problem1. Some studies suggest that MyD88 knockout mice reduce motor deterioration while others demonstrated the involvement of both pathways1. Further investigation is required for a complete understanding of MyD88-dependant and independent mechanism and its involvement in the induction of CNS inflammation 11,12.

Recent findings have also revealed that TLR4 deficient mice are less inclined to ingest alcohol than their WT counterpart and display fewer cognitive and behavioural deficiencies that may be induced by alcohol consumption13. By combining the evidence that TLR4 deficient mice induce less CNS inflammation, these findings support the connection between TLR4 activated CNS inflammation and its respective behavioural impairments. Epigenetic modifications have been demonstrated in WT mice but absent in TLR4 deficient mice, indicating that alcohol consumption may lead to chromatin modifications involving a TLR-mediated mechanism1. This can be the mouse cortex and hippocampus in terms of a decrease in histone acetylation, which may also play a role in behavioural and cognitive dysfunction11.

1.3 Therapeutic Potential

Although there is no cure for FASD, PPAR-y agonists have been shown to suppress several autoimmune diseases in animal models including multiple sclerosis which is described by CNS-wide inflammation1. These drugs have shown to reduce inflammation inducing molecules produced mainly by microglia as well as defend neurons from the toxicity of alcohol2. PPAR-y agonists work by way of reducing gene expression of inflammatory molecules through trans-repression of PPAR-y, associating it with other transcription factors which inevitably inhibit its function in activating inflammatory molecules2. Since PPAR-y is present on neurons, it has been hypothesised that that agonist may prevent neuronal apoptosis by modifying the expression of Bc-2, and anti-apoptosis factor. Furthermore, these agonists induce the translocation of Bax, an apoptotic inducing factor, to the mitochondrial membrane and prevent its further effects2. The protective effects of PPAR-y agonists have also been shown in primary cultures involving microglia and neurons, which suggest that it may be an effective drug in reducing alcohol-induce pathology in the CNS2. However, further research is needed to understand its underlying mechanisms so that a logical path to a cure can be found2.

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